Studies were initiated to define the genetic basis of the temperature-sensitive (ts), cold-adaptation (ca) and attenuation (att) phenotypes of the PIV3 cp45 live attenuated vaccine candidate, which contains 21 mutations, three of which are amino acid (aa) substitutions in the L polymerase protein. To study the individual and aggregate contributions that these three L aa substitutions make to the ts , att and ca phenotypes of cp45, seven PIV3 recombinant viruses (three single-, three double-, and one triple-lesioned virus) representing all possible combinations of the three L aa substitutions were recovered from full-length antigenomic cDNA and analyzed for their ts, att and ca phenotypes. The three individual mutations in L each contributed to the ts and att, but not the ca, phenotype, and this likely contributes to the observed stability of these phenotypes during replication of cp45 in vivo. Importantly, the recombinant virus possessing all three of these mutations was as restricted in replication as the cp45 mutant in both the upper and lower respiratory tract of hamsters, indicating that the L gene of the cp45 virus is a major site for attenuating mutations of this candidate vaccine. The recent recovery of human parainfluenza virus type 3 (PIV3) from cDNA, together with the availability of a promising, highly characterized live attenuated PIV3 vaccine virus, suggested a novel strategy for the rapid development of comparable recombinant vaccine viruses for human PIV1 and PIV2. We have created a chimeric recombinant virus in which the two protective antigens, the hemagglutinin-neuraminidase (HN) and fusion (F) envelope glycoproteins, of wild type PIV3 virus were replaced by those of PIV1. The chimeric recombinant virus, designated rPIV3- 1, replicates in simian LLC-MK2 cells and in the respiratory tract of hamsters as efficiently as its PIV1 and PIV3 parents. It should now be possible to rapidly develop a live attenuated PIV1 vaccine by the introduction of known, characterized attenuating mutations present in a live attenuated PIV3 vaccine candidate, such as cp45, into the PIV3-1 cDNA followed by recovery of attenuated derivatives of rPIV3-1. The efficacy of MVA-PIV3 HN and F recombinants, but not that of the cp45 vaccine, is substantially reduced in rhesus monkeys with passively-acquired PIV3 antibodies indicating that the cp45 is a more promising vaccine candidate for use in the very young infant.